Automated cell processing methods, modules, instruments, and systems

• US 10,323,242 B1
• Filed: 02/07/2019
• Issued: 06/18/2019
• Est. Priority Date: 06/30/2017
• Status: Active Grant

• Current Assignee: Inscripta, Inc.
• Original Assignee: Inscripta, Inc.
• Inventors: Masquelier, Don, Belgrader, Phillip, Bernate, Jorge, Gill, Ryan, Ness, Kevin
• Primary Examiner(s): Bowers, Nathan A
• Application Number:
  US16/269,671
• Publication Number:
  US 20190185846A1
• Time in Patent Office: 131 Days
• CPC Class Codes: C12M 23/44 Multiple separable units; Modules
• CPC Class Codes: C12M 33/14 with filters, sieves or membranes
• CPC Class Codes: C12M 35/00 Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87
• CPC Class Codes: C12M 37/04 Seals
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12M 43/00 Combinations of bioreactors or fermenters with other apparatus
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

In an illustrative embodiment, automated multi-module cell editing instruments are provided to automate multiple edits into nucleic acid sequences inside one or more cells.

Claims

1. A method for editing nucleic acids in live cells in an automated stand-alone multi-module cell editing instrument, comprising the steps of:

providing live cells in a receptacle configured to receive the live cells;

providing first nucleic acids to one or more receptacles configured to receive first nucleic acids;

growing the live cells in a growth module to a desired optical density to produce grown live cells;

transforming the grown live cells in a transformation module configured to introduce the first nucleic acids into the grown live cells to produce transformed cells;

providing conditions in a nuclease-directed editing module to allow the first nucleic acids to edit nucleic acids in the transformed cells to produce edited cells; and

using an automated liquid handling system to

     1) transfer the first nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the live cells from the receptacle configured to receive the live cells to the growth module,

     3) transfer the grown live cells from the growth module to the transformation module, and

     4) transfer the transformed cells from the transformation module to the nuclease-directed editing module without user intervention.

2. The method of editing live cells according to claim 1, further comprising the step of assembling nucleic acids in a nucleic acid assembly module.

3. The method of editing live cells according to claim 1, wherein the transformation module comprises a flow-through electroporation device.

4. The method of editing live cells according to claim 1, wherein the growth module comprises a rotating growth vial.

5. The method of editing live cells according to claim 1, wherein the growth module further comprises a Peltier device.

6. The method of editing live cells according to claim 1, wherein the receptacles for the cells and nucleic acids reside within a reagent cartridge.

7. The method of editing live cells according to claim 6, wherein the reagent cartridge further comprises a flow-though electroporation device.

8. The method of editing live cells according to claim 1, wherein providing conditions in the nuclease-directed editing module to allow the first nucleic acids to edit nucleic acids in the transformed cells to produce edited cells comprises the step of inducing transcription of a nuclease or a gRNA.

9. The method of editing live cells according to claim 1, further comprising the steps of

providing second nucleic acids to one or more receptacles configured to receive nucleic acids;

providing the edited cells from the nuclease-directed editing module to the growth module;

growing the edited cells in the growth module to a desired optical density to produce grown edited cells;

transforming the grown edited cells in the transformation module configured to introduce the second nucleic acids into the grown edited cells to produce transformed edited cells;

providing conditions in the nuclease-directed editing module to allow the second nucleic acids to edit nucleic acids in the transformed edited cells to produce twice-edited cells; and

using an automated liquid handling system to

     1) transfer the second nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the edited cells from the nuclease-directed editing module to the growth module,

     3) transfer the grown edited cells from the growth module to the transformation module, and

     4) transfer the transformed edited cells from the transformation module to the nuclease-directed editing module without user intervention.

10. The method of editing live cells according to claim 9, wherein providing conditions in the nuclease-directed editing module to allow the first nucleic acids to edit nucleic acids in the transformed cells to produce edited cells comprises the step of inducing transcription of a nuclease or a gRNA.

11. The method of editing live cells according to claim 9, further comprising the steps of

providing third nucleic acids to one or more receptacles configured to receive nucleic acids;

providing the edited cells from the nuclease-directed editing module to the growth module;

growing the edited cells in the growth module to a desired optical density to produce grown edited cells;

transforming the grown edited cells in the transformation module configured to introduce the third nucleic acids into the grown edited cells to produce transformed edited cells;

providing conditions in the nuclease-directed editing module to allow the third nucleic acids to edit nucleic acids in the transformed edited cells to produce thrice-edited cells; and

using an automated liquid handling system to

     1) transfer the third nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the edited cells from the nuclease-directed editing module to the growth module,

     3) transfer the grown edited cells from the growth module to the transformation module, and

     4) transfer the transformed edited cells from the transformation module to the nuclease-directed editing module without user intervention.

12. The method of editing live cells according to claim 11, wherein providing conditions in the nuclease-directed editing module to allow the first nucleic acids to edit nucleic acids in the transformed cells to produce edited cells comprises the step of inducing transcription of a nuclease or a gRNA.